Combating intestinal failure with engineered transplantable mucosal grafts

Failure of intestinal function occurs when illness or injury of the small intestine prevents absorption of food and drink, leading to malnutrition and dehydration. It is rare, affecting 1 in 20,000 people, but is incurable and can be fatal. A common cause of irreversible intestinal failure is “short bowel syndrome”, where large sections of the intestine are either congenitally defective or absent, or have had to be surgically removed due to disease. Patients, who frequently include neonates and children, require long term intravenous nutrition, and in the most severe cases, intestinal transplantation may be necessary. Due to a shortage of donor organs and high complication rates, survival rates are low; moreover, even successfully transplanted patients require lifelong immunosuppression to prevent organ rejection.

Given the highly unsatisfactory nature of current treatments, researchers are focussing on developing methods for functional bowel reconstruction through autologous tissue engineering. In a jointly-led study, Vivian Li and Paola Bonfanti of the Crick and Paolo De Coppi of Great Ormond Street Hospital have now moved an important step closer to achieving this aim, showing in a proof-of-concept paper that hybrid small-intestinal grafts integrating paediatric patient-derived intestinal organoids into patient-derived intestinal scaffolds are both functional and durable (Meran et al, 2020).

The group began by establishing a “living biobank” of human paediatric organoids from the duodenum, ileum and jejunum, with particular focus on the jejunum, as this region of the small intestine is where 90% of nutrient absorption occurs. The process was technically challenging, as it depended on tiny endoscopic endothelial biopsies from children with intestinal failure, but a robust protocol was developed for making organoids, which could be expanded to over ten million cells in eight weeks — a number estimated to be sufficient to seed a 20 cm length of tubular scaffold. The organoids could functionally differentiate into various cell types, whose identity depended on which region of the gut they originated from. All organoids retained region-specific marker expression after passaging, demonstrating that their regionality is intrinsically programmed and highly stable in in vitro culture.

In parallel with this work, biological scaffolds derived from the small intestine or colon of paediatric patients undergoing resections were generated and characterised. The scaffolds were decellularised by washing with an enzyme-detergent mix, leaving behind translucent intact scaffolds comprising extracellular matrix components analogous to those of the human intestine. Scanning electron microscopy confirmed that the ultrastructure was conserved after decellularisation: the distinctive mucosal, submucosal and muscle layers were highly preserved, with acellular villi and crypt units visible in the small intestine, and crypts seen in the colon. Investigation of the mechanical properties of the scaffolds showed they were both flexible and durable, and as the biomolecular composition of both small intestine and colon scaffolds was remarkably similar, both types were used in graft development.

Decellularised scaffolds were populated with paediatric jejunal organoids and fibroblasts, and the hybrid grafts were cultured on rafts in a custom-made bioreactor. It was possible to engineer intestinal grafts with a continuous polarised monolayer of columnar epithelial cells covering the entire scaffold, with visible crypt formation, and abundant fibroblasts located subepithelially. The epithelial cells could differentiate into a variety of jejunum-specific cell types, and enterocytes, the primary cells required for nutrient absorption, were detected. The presence of newly deposited collagen underneath the epithelial cells indicated that the transplanted epithelial cells were able to remodel the extracellular matrix. Marker expression showed that jejunal regional identity was preserved after engineering and transplantation, regardless of scaffold origin. The grafts were able to absorb and digest peptides and sucrose, and had good barrier function.

When transplanted subrenally or subcutaneously into immunodeficient mice, the jejunal grafts survived and formed luminal structures. Interestingly, treatment with the drug teduglutide, used in clinics to promote regeneration of the small intestine in patients with intestinal failure, supported the survival of epithelial cells. Adding in endothelial cells also allowed some xenografts to form vasculature, suggesting the possibility that multi-cellular components can self-organise into an even more complex organ.

While it remains to be determined whether the engineered grafts can function as an actual intestine in vivo, this pioneering work is a substantial advance towards the clinical translation of regenerative medicine research into the treatment of patients with intestinal failure. Li and De Coppi plan to scale up the jejunal mucosal grafts for transplantation and validation in larger animal models, and also hope to develop more sophisticated iterations incorporating other components of the gut wall, such as neurons and immune cells. Lastly, such grafts are important tools for research into intestinal development and colorectal cancer, a long-term interest of the Li laboratory, as they have the potential to provide an accessible, physiologically accurate in vitro model for targeted manipulation of key molecules.